Ma et al. BMC Genomics (2021) 22:533 https://doi.org/10.1186/s12864-021-07821-w

RESEARCH Open Access The BBX family in Moso ( edulis): identification, characterization and expression profiles Ruifang Ma1,2, Jialu Chen1,2, Bin Huang1,2, Zhinuo Huang1,2 and Zhijun Zhang1,2*

Abstract Background: The BBX (B-box) family are (ZFP) transcription factors that play an essential role in growth, development and response to abiotic stresses. Although BBX have been characterized in many model organisms, genome-wide identification of the BBX family genes have not yet been reported in Moso bamboo (Phyllostachys edulis), and the biological functions of this family remain unknown. Result: In the present study, we identified 27 BBX genes in the genome of Moso bamboo, and analysis of their conserved motifs and multiple sequence alignments revealed that they all shared highly similar structures. Additionally, phylogenetic and homology analyses indicated that PeBBX genes were divided into three clusters, with whole-genome duplication (WGD) events having facilitated the expansion of this gene family. Light-responsive and stress-related cis-elements were identified by analyzing cis-elements in the promoters of all PeBBX genes. Short time-series expression miner (STEM) analysis revealed that the PeBBX genes had spatiotemporal-specific expression patterns and were likely involved in the growth and development of bamboo shoots. We further explored the downstream target genes of PeBBXs, and GO/KEGG enrichment analysis predicted multiple functions of BBX target genes, including those encoding enzymes involved in plant photosynthesis, pyruvate metabolism and glycolysis/ gluconeogenesis. Conclusions: In conclusion, we analyzed the PeBBX genes at multiple different levels, which will contribute to further studies of the BBX family and provide valuable information for the functional validation of this family. Keywords: Moso bamboo, Gene family, Expression analysis, Characterization

Background domains, oligomerization sites, nuclear localization sig- Transcription factors (TFs) are essential regulatory pro- nals and DNA binding domains [2–4]. Zinc finger TF teins that can activate or repress the expression of their members are divided into several subfamilies, based on target genes and thus play an important regulatory role their structural and functional characteristics. Among in all eukaryotes [1]. This regulation is achieved through them, B-box (BBX) zinc finger have received specific interactions between the DNA-binding domain considerable attention in recent years due to their of the TF and the promoter of the target gene. These diverse functions [5]. interactions are affected by TF transcriptional activation BBX are a class of zinc finger protein TFs with one or two BBX structural domains, with some members also * Correspondence: [email protected] possessing CCT (conserved carboxy-terminal) structural 1State Key Laboratory of Subtropical Forest Cultivation, Zhejiang A&F domains. The most distinctive feature of BBX TFs is the University, Lin’an, Zhejiang 311300 Hangzhou, China 2School of Forestry and Biotechnology, Zhejiang A&F University, Lin’an, presence of one BBX at the N-terminus or two BBX Zhejiang 311300 Hangzhou, China modules in tandem, sometimes with a CCT

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(CONSTANS, CCMike and TOC) domain, at the C- The development and distribution of Moso bamboo terminus [6]. The BBX structure consists of 1–2 BBX are limited by unfavorable climatic and environmental modules with a length of 40 amino acid residues. Based conditions [29]. Genomic studies have the potential to on the similar sequences of the two modules, and the result in dramatic increases in plant stress tolerance spacing characteristics with zinc-binding residues, BBXs [30], but the previously published genome of Moso bam- can be classified into two types: BBX l (B1) and BBX 2 boo has not been sufficiently explored for this purpose. (B2). The zinc finger type of B1 is C-X2-C-X7-8-C-X2- The new assembly of the Moso bamboo genome offers D-X-A-X-L-C-X2-C- D-X3-HB, while the zinc finger an opportunity to improve our understanding of the type of B2 is C-X2-C-X3-P-X4-C-X2-D-X3-L-C-X2-C- abundance, distribution and expansion of bamboo BBX D-X3-H [7, 8]. genes [31, 32]. In this study, we identified 27 genes en- The BBX proteins have unique tertiary structures that coding of Moso bamboo BBX family (PeBBX) and inves- are stabilized by binding Zn ions [9]. Studies have shown tigated their structural domains, amplification patterns, that the BBX domain regulates protein-protein interac- and evolutionary relationships. tions as well as transcriptional processes [10–12]. BBX proteins have been shown to affect seedling development Results by integrating plant phytochromes and light signals Identification and characterization of PeBBX genes in P. sensed by cryptic photoreceptors through the COP1 and edulis HY5 signaling pathways. In addition, some BBX proteins Putative BBX candidate genes were obtained from an are involved in the photoperiodic pathway of flowering HMMER3 search of the bamboo protein database using [13, 14]. Sequence alignment analysis of BBX proteins the plant BBX-type model (Pfam PF00643) with an E- − suggests that the CCT structural domain of some BBX value threshold of ≤ 10 5. We removed redundant genes proteins are also highly conserved and functional [6], and verified the presence of conserved domains and mo- typically consisting of 42–43 amino acids [15]. Both Ara- tifs to arrive at a final set of 27 BBX family members. bidopsis and rice each possess 17 members that have a The genes were renamed PeBBX01–PeBBX27 based on CCT domain at their C-terminus, which plays a vital their positions on chromosomal scaffolds (Table 1). Pro- role in transcriptional regulation and nuclear protein teins encoded by the 27 PeBBX genes contained 70 transport [16, 17]. The Arabidopsis regulator of photo- (PeBBX09) to 446 (PeBBX17) amino acids, and their periodic flowering CO (CONSTAVS), or BBX1, regulates MWs ranged from 7165.31 (PeBBX09) to 48231.99 FT (FLOWERING T) expression to control the (PeBBX17). Their predicted pIs ranged from 4.91 flowering pathway by acting directly on the promoter of (PeBBX04/PeBBX13) to 8.49 (PeBBX15). Aliphatic amino FT [15]. In addition, the CCT structural domain in- acid indices showed that the thermal stability of the pro- cludes nuclear localization signals, which play an active teins ranged from 55.59 to 84.00, indicating that differ- role in the cytosolic localization of BBX TFs [18, 16, 19]. ences in their thermal stability were relatively minor. adapt to various environmental changes by alter- The grand average of hydropathicity (GRAVY) scores of ing their gene regulatory networks [20]. BBX TFs are in- all BBX proteins were negative, suggesting that they duced in response to different abiotic stresses and are were hydrophilic proteins. We predicted the subcellular important candidates for enhancing abiotic stress toler- localization of the proteins encoded by the PeBBX genes ance in plants. In Arabidopsis, COLI (BBX2) is consist- and found that all were likely to localize to the nucleus. ently up-regulated during long periods of cold acclimation [21]andBBX32 in Arabidopsis is responsive to methyl jas- monate (MeJA) hormone stress. In chrysanthemum, Phylogenetic analysis CmBBX24 improves tolerance to abiotic stresses, espe- Previous studies have shown that OsBBX genes can be cially drought stress and low-temperature stress [22]. In divided into five subfamilies [9]. A NJ phylogenetic tree banana, transcripts of the MaCOL1 gene were shown to was constructed based on BBX protein sequences from accumulate rapidly under cold treatment [23]. bamboo, rice and A. thaliana to demonstrate the phylo- Bamboo is one of the most important non-timber for- genetic relationships between Moso bamboo and Grami- est products globally [24]. Moso bamboo (Phyllostachys neae plants as well as dicotyledonous plants (Additional edulis, P. edulis) is fast-growing, easy to establish, and file 4: Table S1). The sequences of A. thaliana were has both ecological and social benefits [25]. Due to these found to be distributed in subclade I, subclade II and beneficial features, bamboo is widely used for , subclade IV. However, in subclade III and subclade V, paper, artwork and food [26, 27]. In China, several spe- only sequences of A. thaliana and rice were found. In cies of bamboo are grown for dual use, with shoots be- subclade IV, Moso bamboo had the highest number of ing harvested for consumption while mature wood is PeBBX sequences, while subclade I and subclade II con- used for timber [28]. tained five PeBBX sequences. Moreover, the BBX of Ma ta.BCGenomics BMC al. et

Table 1 Physicochemical properties of PeBBXs genes ID ID Deduced amino Chromosomal Molecular Theoretical Aliphatic Grand average of hydropathicity Signal Subcellular Rename acids locus weight pI index (GRAVY) Peptide Localization Prediction PH02Gene35355.t1 PeBBX01 211 scaffold3 23327.18 5.9 62.84 -0.632 No Nucleus (2021)22:533 PH02Gene27822.t1 PeBBX02 327 scaffold3 34238.22 5.22 71.01 -0.176 No Nucleus PH02Gene39681.t1 PeBBX03 259 scaffold3 27821.24 5.7 65.33 -0.235 No Nucleus PH02Gene16459.t1 PeBBX04 274 scaffold3 29368.37 4.91 69.23 -0.319 Yes Nucleus PH02Gene45949.t1 PeBBX05 439 scaffold3 47906.54 5.96 66.31 -0.566 No Nucleus PH02Gene19468.t1 PeBBX06 369 scaffold6 39699.18 4.95 70.11 -0.390 No Nucleus PH02Gene05295.t1 PeBBX07 418 scaffold6 45562.27 5.87 65.45 -0.561 No Nucleus PH02Gene19765.t2 PeBBX08 284 scaffold6 29897.52 5.33 75.46 -0.250 No Nucleus PH02Gene25549.t1 PeBBX09 70 scaffold8 7165.31 5.47 84.00 0.137 No Nucleus PH02Gene21910.t1 PeBBX10 382 scaffold8 41886.71 5.05 67.49 -0.410 No Nucleus PH02Gene02845.t1 PeBBX11 272 scaffold8 28909.61 5.27 76.58 -0.196 No Nucleus PH02Gene42827.t1 PeBBX12 321 scaffold9 34325.22 5.29 56.07 -0.429 No Nucleus PH02Gene23282.t1 PeBBX13 212 scaffold12 21867.18 4.91 70.47 -0.228 No Nucleus PH02Gene32457.t1 PeBBX14 346 scaffold14 36826.24 5.64 62.17 -0.294 No Nucleus PH02Gene09921.t1 PeBBX15 393 scaffold15 43019.72 8.49 65.67 -0.557 No Nucleus PH02Gene20422.t1 PeBBX16 344 scaffold16 36537.83 5.33 61.72 -0.328 No Nucleus PH02Gene08112.t1 PeBBX17 446 scaffold17 48231.99 7.05 67.26 -0.551 No Nucleus PH02Gene29322.t1 PeBBX18 261 scaffold17 27900.11 5.26 62.99 -0.306 No Nucleus PH02Gene28332.t1 PeBBX19 326 scaffold17 34492.53 5.25 70.37 -0.271 No Nucleus PH02Gene14035.t1 PeBBX20 257 scaffold17 27748.19 5.06 75.68 -0.295 No Nucleus PH02Gene08231.t1 PeBBX21 322 scaffold18 34437.41 5.65 55.59 -0.457 No Nucleus PH02Gene13905.t1 PeBBX22 394 scaffold21 43179.69 5.94 66.50 -0.589 No Nucleus PH02Gene35510.t1 PeBBX23 249 scaffold23 26905.87 5.28 64.38 -0.427 No Nucleus PH02Gene06669.t1 PeBBX24 332 scaffold23 34866.85 5.25 65.57 -0.308 No Nucleus PH02Gene11378.t1 PeBBX25 256 scaffold23 27484.9 5.01 74.77 -0.286 No Nucleus PH02Gene03202.t1 PeBBX26 255 scaffold24 27165.64 4.98 77.02 -0.177 No Nucleus

PH02Gene02031.t1 PeBBX27 252 scaffold24 27505.72 5.04 67.86 -0.378 No Nucleus 20 of 3 Page Ma et al. BMC Genomics (2021) 22:533 Page 4 of 20

Moso bamboo and the BBX of rice clustered in one a short overall length and only one intron. After com- group (Fig. 1) and were more closely related. paring the motifs of subclades I, II and IV, we found that motif 1 of subfamily IV and subfamily II was in the more Gene structure, conserved domains, motifs and sequence N-terminal position, while motif 2 of subfamily I was in analysis the more N-terminal position (Fig. 2b). After combining To gain insight into the structural features of PeBBX these results with the analysis of conserved structural genes in Moso bamboo, we compared their genomic domains (Fig. 2c), it is clear that motif 3 formed the DNA sequences to determine the number of introns and CCT structural domain, and motif 1 formed the BOX1 exons within each gene. The gene structures of all 27 structural domain. In contrast, motif 2 formed BOX2 PeBBX genes were shown in Fig. 2a. Subclade I and sub- structural domain, and the BOX structural domain was clade II were found to have similar gene structures, with composed of motif 1 and motif 2 together.

Fig. 1 Phylogenetic tree analysis of BBX sequences. The full-length amino acid sequences of 89 BBX proteins were used to construct the phylogenetic tree using MEGA7.0 with the neighbor-joining (NJ) method. The number at the branch represents the confidence value obtained by 100 bootstrap tests. AtBBX represents BBX protein sequence of Arabidopsis thaliana and OsBBX represents BBX protein sequence of rice Ma et al. BMC Genomics (2021) 22:533 Page 5 of 20

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Fig. 2 Gene structure, motifs, conserved structural domains, compositions and multiple sequence alignment analysis of the PeBBX gene family. a. Intron-exon structure of the PeBBX genes. Yellow boxes represent exons (CDS), black lines represent introns and green boxes represent the 5’ and 3’ untranslated regions. b. Distribution of conserved motifs in PeBBX proteins. The scale bar at the bottom indicates the protein lengths, and sequence logos for each conserved motif are shown on the right. c. Conserved domain predictions for the 27 PeBBX proteins. The length of each protein sequence is represented by the grey bars, and colored boxes represent conserved domains. Green boxes represent CCT domains, light purple boxes indicate BOX1 domains and yellow boxes indicate BOX2 domains. d Multiple sequence alignment of PeBBX protein sequences. The number above the sequence indicates the location of the amino acids in the BBX proteins. The key domains are highlighted, and the domain names are shown at the top of the sequence. Protein sequence names are shown on the left

To analyze the sequence characteristics of PeBBX pro- Chromosomal location and gene duplication of PeBBX teins, we used the CDD website to predict the structural genes domains in the full-length sequence of PeBBX proteins, The PeBBX genes were unequally distributed across the revealing that the subclade IV had 17 members, nearly 11 scaffolds of Moso bamboo (Additional all of which had two BOX structural domains (BOX1 file 1: Figure S1). The largest number was found on scaf- and BOX2). PeBBX09 was the only protein sequence in fold 3 (5), followed by scaffold 17 (4), scaffolds 6 (3), 8 subclade IV that had only one BOX1 structural domain. (3), 23 (3) and 2 PeBBX genes were mapped on scaffold In addition, the PeBBX sequences of the subclade II all 24. All other contained a single PeBBX possessed one BOX structural domain and one CCT gene. In all species, gene duplication events are preva- structural domain. In contrast, the PeBBX sequences of lent, and they can produce new functional genes and the subclade IV all contained two BOX structural do- drive the evolution of species. Therefore, we used mains and one CCT structural domain, with the excep- MCScanX genomic co-frequency analysis to explore du- tion of PeBBX04. plications within the BBX gene family (Fig. 3a), revealing To investigate the prevalence and locations of con- 30 segmental duplications in in the whole genome of served protein domains, we created a multiple sequence Moso bamboo. alignment of 27 PeBBX proteins. At the N-terminus, all Monocotyledonous plants, including rice and maize, BBX family members contained a highly conserved BOX and herbaceous plants, including pepper and Arabidop- domain that consisted of approximately 30 amino acids sis, were selected for interspecific collinearity analysis by (Fig. 2d). Some of the BBX sequences also had a CCT BLAST comparison of BBX homologous sequences. This domain at the C-terminus. analysis revealed that the BBX genes in Moso bamboo Ma et al. BMC Genomics (2021) 22:533 Page 6 of 20

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Fig. 3 (See legend on next page.) Ma et al. BMC Genomics (2021) 22:533 Page 7 of 20

(See figure on previous page.) Fig. 3 Chromosomal location and duplicated genes among PeBBX genes. a Intraspecific colinearity analysis. A total of 27 PeBBXs were mapped onto the chromosomes on the basis of their physical location. Chromosome numbers (scaffold1- scaffold24) are distributed in the outer circle, the blue lines indicate duplicated PeBBX gene pairs. b Analysis of collinearity between different species. The gray lines indicate duplicated blocks, while the red lines indicate duplicated BBX gene pairs. Chromosome numbers are at the bottom of each chromosome

and rice were highly homologous, indicating that they bamboo, and found that among these 24 pairs of most likely had similar functions. In the model plant A. homologous genes, PeBBX04/PeBBX10 had the lowest thaliana, no homology was identified with the BBX Ka/Ks ratio (0.1734), followed by PeBBX19/PeBBX02 genes of Moso bamboo. In addition, many highly hom- (0.2422), while PeBBX20/PeBBX25 had the highest ra- ologous genes of BBX in Moso bamboo were identified tio (0.4798). The Ka/Ks ratio of all Moso bamboo in maize, and three homologous genes were identified in BBX homologs were less than 1, indicating a strong pepper (Fig. 3b). purifying selection in the evolution of the Moso bam- Based on the comparative sequence similarity of boo BBX gene family (Table 2).TheKsvaluesob- BBX family, among which 24 Moso bamboo homolo- tained during this analysis then used to calculate the gous gene pairs were found, Ka/Ks ratios of the 24 duplication time of Moso bamboo BBX genes during Moso bamboo BBX gene pairs were calculated, and evolution. This analysis results showed that the repli- the Ks values were used to calculate the time of cation time of Moso bamboo BBX genes primarily duplication events of Moso bamboo BBX genes. We occurred between 9 and 75 million years ago (mya), performed Ka/Ks (evolutionary selection pressure) with nine gene pair replication events occurring in analysis on 24 PeBBX homologous gene pairs in Moso roughly 9–17 mya.

Table 2 Ka/Ks values of homologous PeBBX gene pairs Duplicate gene pair Ka Ks Ka/Ks Purify Selection Duplication type Time (Myaa) PeBBX14/PeBBX16 0.042104596 0.152056047 0.276901819 Yes segmental 11.69661899 PeBBX14/PeBBX21 0.19628168 0.448756769 0.437389905 Yes segmental 34.51975143 PeBBX14/PeBBX12 0.182756627 0.446713424 0.4091138 Yes segmental 34.36257111 PeBBX15/PeBBX22 0.052573616 0.202958669 0.259036072 Yes segmental 15.6122053 PeBBX16/PeBBX21 0.176648673 0.441908756 0.399740151 Yes segmental 33.9929812 PeBBX16/PeBBX12 0.173011971 0.46856959 0.369234315 Yes segmental 36.04381459 PeBBX17/PeBBX07 0.216882382 0.658188855 0.329513908 Yes segmental 50.62991194 PeBBX18/PeBBX23 0.170545595 0.379913416 0.448906482 Yes segmental 29.22410896 PeBBX19/PeBBX24 0.165046414 0.522000187 0.316180756 Yes segmental 40.15386055 PeBBX20/PeBBX25 0.13529533 0.281953784 0.479849316 Yes segmental 21.68875262 PeBBX17/PeBBX05 0.054743612 0.179281304 0.305350367 Yes segmental 13.79086958 PeBBX18/PeBBX03 0.041517811 0.139739347 0.297108955 Yes segmental 10.74918051 PeBBX19/PeBBX02 0.043371875 0.179063587 0.242214932 Yes segmental 13.77412206 PeBBX18/PeBBX27 0.139056615 0.335375829 0.414629209 Yes segmental 25.79814069 PeBBX20/PeBBX26 0.136471916 0.295718856 0.461492101 Yes segmental 22.74760428 PeBBX21/PeBBX12 0.040608208 0.131673581 0.308400577 Yes segmental 10.12873698 PeBBX27/PeBBX03 0.141112234 0.352596186 0.40020919 Yes segmental 27.12278352 PeBBX24/PeBBX02 0.167787595 0.475482406 0.35287866 Yes segmental 36.57556972 PeBBX23/PeBBX03 0.165269986 0.429437165 0.384852546 Yes segmental 33.03362806 PeBBX23/PeBBX27 0.051435606 0.126234197 0.40746174 Yes segmental 9.710322865 PeBBX06/PeBBX09 0.067830454 0.194146225 0.349378174 Yes segmental 14.93432503 PeBBX08/PeBBX11 0.056814836 0.148800797 0.381818089 Yes segmental 11.44621513 PeBBX05/PeBBX07 0.205355044 0.547240162 0.375255799 Yes segmental 42.09539711 PeBBX04/PeBBX10 0.16485542 0.950757381 0.173393784 Yes segmental 73.13518318 amillion years ago Ma et al. BMC Genomics (2021) 22:533 Page 8 of 20

Cis-element analysis of PeBBXs Transcript expression pattern analysis Sequence analysis of the promoter regions of PeBBX genes next quantified the expression patterns of the 27 PeBBX via the PlantCARE website showed 431 associated cis-act- genes at different shoot heights using RNA-seq (Fig. 5, ing elements (Fig. 4). These included 152 cis-elements re- Additional file 6: Table S3). This analysis revealed that lated to light response regulation. In addition, there were all 27 members had different expression patterns. hormone-responsive elements, including Ethylene re- PeBBX16 was highly expressed ubiquitously, while sponse element (ERE), abscisic acid (ABA) responsive ele- PeBBX21, PeBBX26, PeBBX08 and PeBBX27 were highly ments (ABRE, ABA responsiveness) and MeJA acid expressed at 0.2 m. PeBBX19 and PeBBX24 showed a de- responsive elements (CGTCA-motif and TGACG-motif). creasing trend in expression at 4 m. PeBBX17, PeBBX20 There were also other stress responsive elements, anaer- and PeBBX25 showed a positive correlation between obic response regulatory elements (ARE, anaerobic re- their expression levels and the growth height of the sponsiveness), drought-related regulatory elements (MBS), shoots. The gene expression results showed that the low-temperature response elements (LTR, low- PeBBX genes were expressed at all stages of shoot temperature responsiveness), defense and stress respon- development. siveness (TC-rich motif) and wounding and pathogen re- STEM is a measurement of gene expression in similar sponsiveness (W-box). It was noteworthy that each gene samples at different time points to observe the changes had a unique composition of cis-acting elements (Add- in gene expression at each time point and elucidate the itional file 2: Figure S2, Additional file 5: Table S2), with patterns of changes in interdependent relationships be- PeBBX06, PeBBX13 and PeBBX14 containing more light- tween genes. Temporal sequence analysis based on responsive cis-elements, while PeBBX06 and PeBBX14 STEM was applied to the gene expression patterns of contained more hormone-responsive elements. PeBBX genes at multiple heights during the growth of

Fig. 4 Cis-element analysis of PeBBXs. Information about the promoters of PeBBX genes. The graph on the left shows cis-acting element enrichment, with different colors indicating different element numbers. The chart on the right is a proportional map, the blue rectangle represents light responsive cis-elements, the orange rectangle represents hormone responsive cis-elements, and the gray rectangle represents stress responsive cis-elements Ma et al. BMC Genomics (2021) 22:533 Page 9 of 20

Fig. 5 Analysis of the expression patterns of different heights of bamboo shoots. That three replicates per condition. The relative expression levels are depicted according to the colour scale, where blue indicates low abundance and red indicates high abundance. Gene expression log2 (TPM) was calculated as the sum of the abundance of all transcripts produced by a given gene shoots, revealing 10 different gene expression profiles predicted to interact with each other (Fig. 7). The (Fig. 6a). Profile 9 contained 7 PeBBX genes, which all PeBBX01 protein had the highest connectivity, interact- had expression levels that were positively correlated with ing with 13 BBX proteins of Moso bamboo. shoot height. Within this profile, PeBBX14, PeBBX15, PeBBX25 and PeBBX26 had the highest expression at PeBBX target gene identification, GO, KEGG annotation 5 m, while PeBBX07 had the highest expression at 4 m and analysis. (Fig. 6b). We predicted BBX target genes using the JASPAR data- base and identified two BBX binding patterns (Fig. 8a), Protein interaction network which were used to predict BBX target genes (Additional A protein interaction network was constructed using a file 8). GO enrichment analysis and KEGG enrichment network modelling method at STRING, its topological analysis of BBX target genes were also performed on GO properties were analyzed at the network level. This ana- enrichment tool (http://geneontology.org/page/go- lysis revealed that 14 proteins in the BBX family were enrichment-analysis) and KEGG website, respectively. Ma et al. BMC Genomics (2021) 22:533 Page 10 of 20

Fig. 6 STEM analysis of PeBBX genes. a STEM analysis of PeBBX genes. The yellow boxes indicate significant gene expression. All 10 profiles are drawn with profile numbers denoted at the top left. The dashed line indicates the trend of expression over time, and the value in the lower-left corner is the P-value for its corresponding significance level. The image on the right shows the manifestations of significantly expressed genes. b Expression patterns of genes in profile 9 that are highly positively associated with shoot growth stages. A single asterisk indicates that the level of the gene expression was significantly different (t-test, p < 0.05). Double asterisks indicate that there is a significant difference (t-test, p < 0.01). Error bars represent standard deviations of the means of three independent replicates

GO annotation enrichment was divided into three levels: PH02Gene42040, both of which are Chlorophyll a-b biological process (BP), cellular component (CC) and binding proteins (CP26). These targets indicated that molecular function (MF). A total of 185 target genes PeBBX genes were associated with the formation of were predicted to be regulated by BBX members and complexes that is involved in the regulation of photosyn- were mainly enriched in categories falling under CC and thesis (Additional file 10). KEGG predicted that the BP (Fig. 8b). At the CC level, targets were primarily major metabolic pathways enriched in BBX target genes enriched in the Golgi apparatus (GO:0005794), and at were found to be primarily involved in Phenylpropanoid the BP level, targets were mainly enriched in response to biosynthesis, Photosynthesis-antenna proteins, Pyruvate oxygen-containing compound (GO:1,901,700) and re- metabolism and Glycolysis/Gluconeogenesis (Fig. 8c, sponse to chemical (GO:0042221). At the MF level, there Additional file 11). was only one enriched GO term: pigment binding (GO: 0031409). In addition, GO predicted that some BBX tar- Protein structure analysis by homology modeling get genes were components of the photosystem com- To further analyze the protein function, the tertiary plexes, including photosystem II (PSII) antenna structures of the proteins were predicted. PeBBX04 from complex, PSII associated light-harvesting complex II, subclade I, PeBBX05 from subclade II and PeBBX13 light-harvesting complex and thylakoid light-harvesting from subclade IV were selected for tertiary structural complex (Additional file 9). The target genes included homology modelling. The 3D structures of the three photopigment PR genes, such as PH02Gene47533 and subclades were found to have different tertiary structures Ma et al. BMC Genomics (2021) 22:533 Page 11 of 20

Fig. 7 Protein interaction network analysis. Nodes indicate proteins, and edges indicate the presence of interactions between two proteins. Nodes in orange indicate the highest level of connectivity for that protein

(Fig. 9). The PeBBX04 sequence consisted of two α- Arabidopsis, 30 in rice, 29 in tomato, 30 in potato and helices and two β-folded lamellae and contained three 37 in pear [2, 9, 6, 35, 36]. amino acid residues that specifically bind to zinc ions In this study, we identified a total of 27 genes encod- (C.88, C.105, C.108). The PeBBX05 sequence consisted ing BBX proteins (PeBBX) in the Moso bamboo genome. mainly of β-folded lamellae, and the PeBBX13 sequence The B-box1 and B-box2 structural domains of the 27 was more complex, with four β-folded lamellae. PeBBX protein sequences were highly conserved, and there was a strong similarity with the BBX sequences of Subcellular localization of BBX proteins rice and Arabidopsis. This indicated that the sequences PeBBX01 showed high connectivity in protein inter- of BBX were strongly conserved during the evolutionary action network prediction, and to further understand the process. The number of genes encoding specific TF fam- properties of this protein, we selected PeBBX01 for sub- ilies varies among plant species due to evolutionary ex- cellular localization analysis. We constructed a transient pansion and the development of species-specific expression MAS-PeBBX01-GFP vector for this gene for functions [37]. The results of the phylogenetic tree validation experimental purposes. The results showed showed that PeBBXs lacks the III and V subclades, and that the MAS-GFP fluorescent signal in the control was expanded the subclade IV compared to Arabidopsis and dispersed throughout the tobacco cells, and the rice. Among the BBX family members of Arabidopsis,21 expressed fusion protein MAS-PeBBX01-GFP was spe- BBX have been functionally characterized. Among them, cifically distributed in the nucleus (Fig. 10). there were eight BBX proteins that were members of subclade IV and had either facilitative or repressive ef- Discussion fects on photomorphogenesis. Recent analysis of BR- Plants are often challenged with various unfavorable en- related (brassinosteroid action-related) genes in bamboo vironmental conditions throughout their growth and de- has shown that the phytohormone BR regulated growth velopment. In response to these environmental changes, by promoting root cell division and elongation [38, 39], a series of physiological and biochemical changes are and ATBBX20 in subcladeIV responded to the phytohor- produced. A diverse array of different genes are required mone BR [14]. In addition, these genes were involved in for these changes, with TFs playing a central role [33, salt, cold, darkness, and hormone responses [14]. 34]. Zinc finger TFs are a superfamily of TFs with many OsBBX14, a member of rice subclade IV. Under photo- members, including BBXs. In plants, several BBX gene periodic conditions, OsBBX14 expression had a circadian families have been identified, with 32 members in rhythm and the OsBBX14 overexpression strain Ma et al. BMC Genomics (2021) 22:533 Page 12 of 20

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Fig. 8 (See legend on next page.) Ma et al. BMC Genomics (2021) 22:533 Page 13 of 20

(See figure on previous page.) Fig. 8 BBX binding pattern, GO and KEGG annotation. a BBX target gene prediction. The two predicted BBX binding patterns resulted in the targeting of two different sets of genes, termed target genes. b GO enrichment analysis of BBX target genes from the enrichment top 20. The vertical axis represents the GO term, and the horizontal axis represents the Rich factor, with larger Rich factor indicating greater enrichment. The size of the dots indicates the number of genes in the GO term, and the color of the dots corresponds to different P-value ranges. The top 20 enrichment results are displayed for P-value < 0.05. c)KEGG enrichment analysis of BBX target genes from the top 20. The vertical axis represents the pathway name, the horizontal axis represents the Rich factor and the size of the points indicates the number of genes in the pathway. The top 20 enrichment results are displayed for P-value < 0.05

(OsBBX14-OX) delayed heading date by repressing the deletion of the 2 BBX and CCT structural domains dur- expression of the flowering-forming factor gene under ing the subsequent evolution, as well as the further repli- long-day (LD) and short-day (SD) conditions [40]. Based cation events of the BBX structural domain, helped BBX on studies in the subclade IV of Arabidopsis and rice, it proteins expand into different structural types [7]. is hypothesized that the expansion of the subclade IV of PeBBX09 had only one BOX1 domain, presumably due PeBBX may have an effect on flowering and photo- to a deletion that occurred during evolution. morphogenesis and development phenomena in Moso Gene duplication has been shown to be a significant bamboo. factor in the evolution of genes with novel functions The motif analysis revealed that different BBX proteins [45]. In the process of species evolution, the duplication in subclades I, II and IV contain motifs with unclear events can promote the evolution of species and the functions. In addition, there were three BBX proteins rapid expansion of the species genome, and most of the (PeBBX02, PeBBX19, PeBBX24) had a valine-proline duplication events occur in species due to the drastic (VP) motif consisting of 6 amino acids at the C- changes in the survival environment [46]. Furthermore, terminus, with a consistent sequence of G-I/V-V-V-P-S/ during the growth and development of species, gene du- T-F (Additional file 3: Figure S3). The VP motif is gener- plication events mainly help species to adapt to different ally about 16–20 amino acid residues away from the environments, thus adapting to the drastically changing CCT structural domain, which plays a vital role in the external environment and increasing the survival rate of interaction between BBX protein and coiled-coil proteins the species [47, 48]. Jiao et al. [49] showed that one of [41, 42]. COP1 (CONSTITUTIVE PHOTOMORPHO- the gene duplication events in plants occurred 192 mya. GENIC 1) is involved in the degradation of HY5 as a In addition, it has been reported that the Moso bamboo pan-peptide ligase component and inhibits plant photo- SPL gene family had a large duplication event at about morphogenesis [43]. Additionally, it was previously 15 mya [26]. Among the gene pairs identified, PeBBX14/ shown that different VP motifs allow different BBXs to PeBBX16, PeBBX17/PeBBX05, PeBBX18/PeBBX03, bind the WD40 (Trp-Asp-40) structural domain of PeBBX19/PeBBX02, PeBBX21/PeBBX12, PeBBX23/ COP1 with different affinities, thereby regulating photo- PeBBX27, PeBBX06/PeBBX09 and PeBBX08/PeBBX11 morphogenesis [44]. occurred between 9 and 15 mya, close to the previously The amino acid sequences of B-box1 and B-box2 reported large-scale duplication events of 7–15 mya for structural domains in animals is significantly different the whole genome of Mao bamboo [26]. compared to plants, but within plants these domains are In gene family evolution and expansion, tandem dupli- much more consistent. In addition, the evolutionary tra- cation and segmental duplication occur frequently [46]. jectory of BBX domains in green plants indicates that Zhang et al.[50] found that fragment duplication was the the original BBX protein had only one BBX domain, main driving force for the expansion of the RPD3 gene which underwent a duplication event later in evolution. family in cotton in a covariance analysis. In the Moso After this event, the CCT domain was formed. The bamboo BBX gene family, no tandem gene clusters were

Fig. 9 Three-dimensional structure of the Moso bamboo BBX protein sequences. Models were constructed with the SWISS-MODEL software. The red areas indicate the α-helices and the bright blue areas represent the β-folds. C88, C105 and C108 indicate cysteines bound to a zinc ion Ma et al. BMC Genomics (2021) 22:533 Page 14 of 20

Fig. 10 Subcellular localization of the GFP-fused PeBBX protein. a Schematic drawing of PeBBX01 subcellular localization. b Subcellular localization of PeBBX01. The fusion protein MAS-PeBBX01-GFP and the control vector were transiently expressed in tobacco leaves and then observed by fluorescence microscopy. Scale bar was 50 μm identified, and 24 gene pairs were identified, suggesting contained drought-responsive cis-elements were affected that segmental duplication events may be more import- by drought in Petunia hybrida [56]. Moso bamboo has ant than tandem duplication in the expansion of the the property of explosive growth, which can grow about PeBBX genes. Interestingly, two genes of these homolo- 20 m in 1.5 months [57]. It was found that the growth of gous gene pairs belonged to the same subclade, suggest- Moso bamboo was correlated with GA. Exogenous gib- ing that some BBX family genes might arise from gene berellin (GA3) was applied to Moso bamboo seedlings duplication events and that these duplication events and it was observed to significantly increase internode were the main drivers of gene family expansion [51]. As length [58]. In the currently study, there were 9 genes a result of interspecies collinearity analysis, some hom- containing ERE promoter elements and 14 genes con- ologous regions were formed between different chromo- tained drought response elements (MBS), and almost all somes. Some PeBBX genes fell within these homologous of them contained ABRE. In addition, a large number of regions, suggesting that genome-wide amplification of light response elements, hormone response elements the BBX gene family also occurred. The expansion of and abiotic stress response elements were distributed in BBX during evolution and their high conservation the promoters of PeBBXs, suggesting that these genes throughout different plant species suggest that BBX may might play essential functions in light signaling, hor- play an essential role in the adaptation of plants to ter- mone and stress response. restrial environments [52, 14]. There were studies have shown that growth regulation Plant hormones were originally described as regulators effect of BBX [59]. AtBBX24 binded to DELLA protein during growth and development, acting as signaling mol- and prevented DELLA-mediated inhibition of PIF4 activ- ecules to regulate complex metabolic pathways [53, 54]. ity, which in turn PIF4 binded to the promoter of cell In a previously study, four tomato BBX genes were in- elongation-related genes to promote cell elongation [60]. duced by ethylene (ETH) and all of them had ERE in It has been shown that expression of apple MdBBX10 in their promoters [2]. In grapes, four BBX genes expres- Escherichia coli enhanced cellular tolerance to salt and sion (VvBBX2, VvBBX3, VvBBX13 and VvBBX22) were osmotic stress, and overexpression of this gene in Arabi- affected by ABA and ETH, and the promoter regions of dopsis also enhanced the tolerance of transgenic plants all four genes contained ABRE promoter elements [55]. to abiotic stresses, such as drought and salt [61, 62]. Three PhBBX genes (PhBBX8, PhBBX15 and PhBBX20) High salt and polyethylene glycol (PEG) treatment- Ma et al. BMC Genomics (2021) 22:533 Page 15 of 20

induced SsBBX24 gene expression and protein accumula- complex that catalyzes light-driven water oxidation and tion in potato (Solanum sogarandinum), and the length of reduction of plastid quinones. The light-trapping chloro- daylight also regulated the response of SsBBX24 to salt phyll a/b binding protein is the light-carrying protein of stress [63]. In addition to direct regulation at the tran- the PS II light-trapping complex. Photons captured by scriptional and protein levels, BBX proteins have been the light-trapping chlorophyll a/b-binding antenna com- shown to play important roles in hormone signaling by plex light-catching complex II (LHCII) are converted by growth hormones, such as indole-3-acetic acid (IAA), gib- photosynthesis into biochemical energy and biomass for berellic acid (GA), ABA and brassinolide (brassinosteroid, regulating plant growth, development and morphogen- BR) [64]. AtBBX21 acts as a negative regulator related to esis [70–73]. growth hormones, ETH and BR, thus affecting plant The phenylpropanoid/flavonoid biosynthesis pathway growth under long-term shade conditions [65]. Addition- is an important metabolic pathway in plants. For ex- ally, GA may be involved in the regulation of flowering in ample, plant defense responses are associated with acti- Dendranthema morifolium by CmBBX24 [22]. Addition- vation of the general phenylpropanoid pathway and ally, our expression analysis indicated that PeBBX genes anthocyanins are the end product of a branch of the might play an important role in the process of plant phenylpropanoid/flavonoid biosynthesis pathway [74, growth and development, different PeBBX genes had ex- 75]. It has been shown that BBX proteins not only play pression specificity at different heights. an essential role in regulating photomorphogenesis [76], Protein interaction networks contribute to the under- but also light-induced anthocyanin synthesis. It was pre- standing of complex biological network systems [66]. Re- viously demonstrated that in Arabidopsis, HY5 is a core cent studies have revealed that the plant BBX structural regulator that facilitates the photomorphogenesis domain plays an important role in mediating protein in- process and that AtBBX32 can bind to AtBBX21 and teractions and regulation of gene expression. In plants, then interact with HY5 to reduce its transcriptional ac- the BBX domain functions by forming heterodimers tivity [77, 78]. In contrast, AtBBX24 might interfere with within the BBX protein family. In Arabidopsis, AtBBX24 the binding of HY5 to the promoter of anthocyanin and AtBBX25 have been shown to interfere with the genes by forming a heterodimer with HY5 [79]. function of HY5 (ELONGATED HYPOCOTYL 5) by PpBBX16 in pear (Pyrus pyrifolia) has also been shown forming an inactive heterodimeric form, which in turn to be a positive regulator of anthocyanin in berries [80]. affected AtBBX22/LZF1 (LIGHT-REGULATED ZINC The enrichment analysis of target genes of BBX were FINGER PROTEIN 1) expression and inhibited the also closely related to the carbohydrate metabolism photomorphogenesis of seedlings [10]. Arabidopsis B- pathway. It has been described previously that carbohy- BOX32 interacts with BBX4/CONSTANS-LIKE3 drates play an important role in plant growth and stress (COL3) to regulate flowering [67]. tolerance [81, 82]. Overall, the analysis of the KEGG In addition, BBX proteins from different plants can metabolic pathway led to a better understanding of the also have cross-species effects. For example, the N- target genes and the pathways regulated by PeBBX. terminal BBX region of AtBBX32 in Arabidopsis has been shown to interact with the BBX protein of Conclusions GmBBX62 in soybean (Glycine )[12]. Protein inter- In this study, 27 members of the Moso bamboo BBX action network analysis speculated that PeBBX proteins family were examined and divided into three subclades interact and PeBBX01 interacted with multiple proteins based on conserved domains. Some fragment duplica- and it might play important roles within the Moso bam- tions were found on different chromosomes, which likely boo family. drove the expansion of the PeBBX gene family. Bioinfor- BBX, as a , can regulate other pro- matic analysis of gene structure, conserved motifs, teins and thus affect plant growth and development. For chromosomal position, covariance and cis-element pre- example, CmBBX8 accelerated flowering in chrysanthe- diction greatly expanded the current understanding of mums by directly targeting CmFLT1, a flower-inducible the structural relationships among family members. Tar- gene [68]. BBX16 promoted the growth of shade hypo- get gene prediction of PeBBX genes resulted in the iden- coty possibly as a positive transcriptional regulator of tification of several key functional genes, opening up a PIL1 [65]. Enrichment analysis of the top 20 BBX target number of new avenues for exploring the role of these genes predicted that many were involved in the photo- genes in Moso bamboo development. synthetic pathway, such as the PSII antenna complex, PSII associated light-harvesting complex II, light- Materials and methods harvesting complex and others. It has been found that Identification of BBX genes in the P. edulis genome PSII is the first complex involved in oxygenic photosyn- The Moso bamboo genome was downloaded from thesis [69]. PSII is a multisubunit pigment-protein GigaDB (http://gigadb.org/dataset/100498). In order to Ma et al. BMC Genomics (2021) 22:533 Page 16 of 20

identify the BBX TF genes in P. edulis, the BBX domain psb.ugent.be/webtools/plantcare/html/), followed by Hidden Markov Model (HMM) was used as a query to functional categorization and visualization using TBtools search the Moso bamboo genome. After removing re- [87]. dundant hits and initial chromosomal localization, BBX family genes were identified by further analysis using CDD (http://www.ncbi.nlm.nih.gov/Structure/cdd/ Chromosomal locations, genomic duplications and Ka/Ks wrpsb.cgi) to reveal potential non-redundant BBX TF ratios genes, termed PeBBX genes. The Molecular weight To identify the locations of the PeBBX members on the (MW) and isoelectric point (pI) of the deduced amino Moso bamboo chromosomes, the termination and start acid sequences were predicted with the ExPASy online positions of the coding sequences (CDSs) were retrieved tool. Next, the SignalP 4.1 Server (http://www.cbs.dtu. from the Moso bamboo database. Each PeBBX gene was dk/services/SignalP/) was used to predict signal peptides placed on the corresponding Moso bamboo chromo- of each member of the BBX family [83, 84]. Finally, some according to the physical location of the gene and Plant-mPLoc was employed to predict the subcellular schematically mapped through the MapChart online localization of proteins [85]. website. Circos software was used to visualize the gene duplications of Moso bamboo PeBBX genes [88]. Se- Phylogenetic analysis quences from rice, Arabidopsis, pepper and maize were The BBX protein sequences of Arabidopsis and rice were identified by BLAST comparison to determine homolo- downloaded from the Arabidopsis Information Resource, gous gene pairs [89]. Then, MCScanX was used to iden- version 10 (TAIR 10) (http://www.arabidopsis.org) and tify homologous regions [90]. the RGAP 7 database (Rice Genome Annotation Project, The ratio of non-synonymous substitutions rate (Ka)/ http://rice.plantbiology.msu.edu/). The BBX protein se- synonymous substitutions rate (Ks) was used to deter- quences from A. thaliana, Oryza sativa and P. edulis mine the homologous relationship and divergence time were used to construct a phylogenetic tree using the of PeBBX genes. For Ka/Ks analysis, 24 homologous neighbor-joining (NJ) method. For statistical reliability, gene pairs were identified by BLAST. Ka/Ks ratios be- the nodes of the tree were assessed by bootstrap analysis tween homologous gene pairs were calculated using containing 1000 replicates. KaKs_Calculator 2.0 [91]. The evolutionary divergence time within thePeBBX genes was computed using the Analysis of gene structure, motifs, domains and multi- bamboo-specific divergence time formula T = Ks/2λ (λ = − sequence alignments 6.5 × 10 9)[24]. To better understand the structures of the PeBBX genes, their exon-intron information was obtained from the whole genome GFF annotation file, then visualized with Expression patterns of PeBBX genes TBtools. To better characterize the structures of the To analyze the expression patterns of the PeBBX genes PeBBX proteins, the software Multiple Expectation and identify genes that may be related to rapid shoot de- Maximization for Motif Elicitation (MEME) was used to velopment, we downloaded RNA-seq data (Accession: identify conserved motifs [86]. The following SRX3282084, SRX3282085, SRX3282086, SRX3282087, optimization parameters were used: any number of re- SRX3282088, SRX3282089, SRX3282090, SRX3282091, peats, optimum width of each motif restricted between 6 SRX3282092, SRX3282093, SRX3282094, SRX3282095, and 50 residues and a maximum number of 5 motifs. SRX3282096, SRX3282097, SRX3282098, SRX3282099, Alignments of protein sequences and the corresponding SRX3282100, SRX3282101, SRX3282102, SRX3282103, residues were carried out using Geneious software with SRX3282104, SRX3282105, SRX3282106, SRX3282107) embedded parameters set to cost matrix: Blosum62. In from the gene expression profile database of NCBI addition, conserved domain analysis was performed (http://www.ncbi.nlm.nih.gov/geo/). Transcriptomic data using Conserved Domain Search (https://www.ncbi.nlm. with replicates came from bamboo shoots at different nih.gov/Structure/cdd/wrpsb.cgi). growth stages (0.2 m, 0.5 m, 1 m, 2 m, 3 m, 5 m, 6 and 7 m). Transcriptome data which was quantified as tran- Cis-acting elements in the PeBBX gene promoter regions scripts per million reads (TPM) were log2-transformed In order to identify conserved cis-elements within the [92] and imported into TBtools, where Amazing Heat- promoter regions of the PeBBX genes, the sequences map was used to generate expression heatmaps. For 1500 bp upstream of the initiation codons were consid- time-series gene expression analysis, we further ered as the proximal promoter region sequences. These employed the STEM algorithm with a number of time- sequences were submitted to the PlantCARE database series patterns of 10 and a significant trend P-value set for promoter prediction analysis (http://bioinformatics. to 0.001. Ma et al. BMC Genomics (2021) 22:533 Page 17 of 20

Protein-protein interaction (PPI) network construction GO mapping analysis and KEGG protein pathway anno- PPI relationships were analyzed using protein sequences tation based on the KEGG database (http://www.kegg.jp/ from PeBBX members. Prediction of PPI networks was kegg/ko.html)[97]. performed using the Retrieval of Interacting Genes/Pro- teins online database (STRING v10) (http://string-db. Protein structure analysis by homology modeling org)[93]. Cytoscape is an open-source bioinformatics The three-dimensional (3D) structures of PeBBX pro- software platform used for visualizing molecular inter- teins were predicted in order to analyze potential protein action networks [94], which we employed to build PPI functions. The tertiary structures of PeBBX proteins network maps. The minimum required score for the were generated by homology modeling techniques using interaction was set to ≥ 0.15 when constructing the the online template-based SWISS-MODEL (http:// interaction network. swissmodel.expasy.org)[98].

Plant Material, RNA extraction and quantitative real-time Subcellular localization of BBX proteins (qRT-PCR) analysis To verify the localization of PeBBX01 in plant cells, the In order to analyze the tissue expression pattern of PeBBX01 gene was selected to design primers with a PeBBX genes, various growth stages samples of Moso homologous recombination arm (the primers see Add- bamboo tissues were collected from bamboo plants itional file 12), and the full-length CDS sequence with growing in the local Moso bamboo forests in Guilin, no terminator codon amplified from the cDNAs of Moso Guangxi (All samples collection with protocols approved bamboo, then the fragment was cloned into the p1300- by the owner), including various young parts of 0.2 m, GFP vector. Within this vector, the PeBBX01 gene was 0.5 m, 1 m, 2 m, 3 m, 5 m, 6 and 7 m. The collected fused with the green fluorescent protein (GFP) gene samples were kept in three independent replicates and under the control of the promoter to form a recombin- all samples were stored at -80 °C until further experi- ant vector. The accuracy of the positive clone was en- ments, followed by grinding to fine powder with a mor- sured by PCR and DNA sequencing, and it was tar and pestle. transformed into the Agrobacterium tumefaciens strain A FastPure Plant Complete RNA Isolation Package GV3101 [99, 100],. Tobaccos (Nicotiana benthamiana) was utilized to extract RNA from Moso bamboo samples were cultivated in a greenhouse at 23 °C for about one ’ for qRT-PCR analysis, following the manufacturer s in- month, then the tobacco in good growth condition was structions (Vazyme company, China). First-strand cDNA selected, and Agrobacterium tumefaciens carrying the re- was synthesized with a HiScript@ lll 1st Strand cDNA combinant vector was injected into the third or fourth Synthesis Kit (+ gDNA wiper), following the manufac- leaf and incubated at 25 °C for 24 h in the dark, followed ’ turer s instruction. Gene-specific qRT-PCR primers were by 24–72 h in the light, to make sections for observation designed by Primer Premier 5 (See Additional file 7: under a laser confocal microscope (Olympus, Tokyo, Table S4) based on the CDS of each gene. Each qRT- Japan)[55] PCR experiment was conducted in triplicate using separ- ate RNA samples. The reaction conditions were: 95 °C Abbreviations for 5 min, followed by 38 cycles of 95 °C for 15 s and TFs: Transcription factors; BBX: B-box; CCT: CONSTANS, CCMike and TOC; BBX 1: B1; BBX 2: B2; CO: CONSTAVS; FT: FLOWERING LOCUS T; MeJA: Methyl 55 °C for 15 s. A melting curve from 65 to 95 °C was jasmonate; ABA: Abscisic acid; ARE: Anaerobic response regulatory elements; then used. The actin gene was used as an internal con- LTR: Low-temperature response elements; BP: Biological process; CC: Cellular trol for normalization. Data were subjected to one-way component; MF: Molecular function; PSII: Photosystem II; VP: Valine-proline; ’ COP1: CONSTITUTIVE PHOTOMORPHOGENIC 1; PEG: Polyethylene glycol; analysis of variance (ANOVA), followed by Tukey s test IAA: Indole-3-acetic acid; GA: Gibberellic acid; BR: Brassinolide using SPSS software [95]. (brassinosteroid); HY5: ELONGATED HYPOCOTYL 5; LZF1: LIGHT-REGULATED ZINC FINGER PROTEIN 1; LHCII: Light-catching complex II; MEME: Multiple PeBBX Expectation Maximization for Motif Elicitation; CDS: Coding sequences; target gene identification, annotation and TPM: Transcripts per million reads; PPI: Protein-protein interaction; qRT- expression analysis PCR: Quantitative real-time; STEM: Short time-series expression miner; Gene promoter sequences (2000 bp upstream of the LD: Long-day; SD: Short-day; ETH: Ethylene; GFP: Green fluorescent protein start site) were extracted from the genome of Moso bamboo. The JASPAR database (http://jaspar.genereg. Supplementary Information net/) was used to predict the binding site genes of The online version contains supplementary material available at https://doi. org/10.1186/s12864-021-07821-w. PeBBX [96]. The screening index was set to less than 1.0 − 6 E to identify the genes targeted by BBX members. Additional file 1: Figure S1. The distribution of the PeBBXgenes on the Geno ontology (GO) annotation was performed using scaffolds of Moso bamboo. PeBBX genes are numbered 1-27. The the UniProt-GOA database (www.http://www.ebi.ac.uk/ chromosome number is shown at the top of each strip, with the gene name displayed on the right or left side of the chromosome. GOA/). Protein IDs were converted to UniProt IDs for Ma et al. BMC Genomics (2021) 22:533 Page 18 of 20

Additional file 2: Figure S2. Location of cis-elements in the promoters land, and all samples collection with protocols approved by the owner. Since of PeBBX genes. Cis-element analysis of PeBBXs. The 1500 bp DNA this study did not involve any endangered or protected species, no ethical fragments upstream of the ATG were analyzed using the online analysis approval or consent was required. software PlantCARE. Different cis-acting elements of PeBBXgenes are displayed. The different colored markers indicate different predictedcis- Consent for publication acting elements. Not applicable. Additional file 3: Figure S3. VP motif. Valine-proline (VP) motif, the proteins with red underlines possess typical VP residues. Competing interests Additional file 4: Table S1. The IDs and sequences of BBX proteins The authors declare no conflict of interest. from rice, Arabidopsisand Moso bamboo used in phylogenetic tree construction. Received: 5 March 2021 Accepted: 17 June 2021 Additional file 5: Table S2. Cis-element analysis of PeBBX genes. Additional file 6: Table S3. The FPKM value of PeBBX genes in different heights. References Additional file 7: Table S4. The primers used for qRT-PCR. 1. Gong W, Shen YP, Ma LG, Pan Y, Du YL, Wang DH, Yang JY, Hu LD, Liu XF, Dong CX, et al. Genome-wide ORFeome cloning and analysis of Arabidopsis Additional file 8. BBX target genes transcription factor genes. Plant Physiol. 2004;135(2):773–82. Additional file 9: The founctional GO analysis. 2. Chu Z, Wang X, Li Y, Yu H, Li J, Lu Y, Li H, Ouyang B. Genomic Organization, Additional file 10: The founctional annotation of BBX target genes. Phylogenetic and Expression Analysis of the B-BOX Gene Family in Tomato. Front Plant Sci. 2016;7:1552. Additional file 11: The founctional KEGG analysis. 3. Smita S, Katiyar A, Chinnusamy V, Pandey DM, Bansal KC. Transcriptional Additional file 12: The primers of PeBBX01. Regulatory Network Analysis of MYB Transcription Factor Family Genes in Rice. Front Plant Sci. 2015;6:1157. 4. Li J, Han G, Sun C, Sui N. Research advances of MYB transcription factors in Acknowledgements plant stress resistance and breeding. Plant Signal Behav. 2019;14(8):1613131. The authors would like to thank TopEdit (www.topeditsci.com) for linguistic 5. Shalmani A, Fan S, Jia P, Li G, Muhammad I, Li Y, Sharif R, Dong F, Zuo X, Li assistance during preparation of this manuscript. Not applicable. K et al. Genome Identification of B-BOX Gene Family Members in Seven Rosaceae Species and Their Expression Analysis in Response to Flower Guidelines or legislation statement Induction in Malus domestica. Molecules 2018;23(7):1763–87. The experimental study of plants, the plant material used and the collection 6. Khanna R, Kronmiller B, Maszle DR, Coupland G, Holm M, Mizuno T, Wu SH. of plant material for this experiment complied with relevant institutional, The Arabidopsis B-box zinc finger family. Plant Cell. 2009;21(11):3416–20. national and international guidelines and legislation. And the study complied 7. Crocco CD, Botto JF. BBX proteins in green plants: insights into their with local and national regulations. evolution, structure, feature and functional diversification. Gene. 2013;531(1): 44–52. Authors' contributions 8. Massiah MA, Matts JA, Short KM, Simmons BN, Singireddy S, Yi Z, Cox TC. RFM was involved in the study design and interpretation, conducted the Solution structure of the MID1 B-box2 CHC(D/C)C(2)H(2) zinc-binding experiments and wrote the manuscript. JLC helped analyze the data. BH and domain: insights into an evolutionarily conserved RING fold. J Mol Biol. ZNH helped revise the manuscript. ZJZ was involved in the design and 2007;369(1):1–10. interpretation of the study and refined this manuscript. ZJZ was responsible 9. Huang J, Zhao X, Weng X, Wang L, Xie W. The rice B-box zinc finger gene for the completeness of the data and the accuracy of the data analysis. All family: genomic identification, characterization, expression profiling and members were involved in the manuscript modification and all authors have diurnal analysis. Plos One. 2012;7(10):e48242. read and agreed to the published version of the manuscript. The author(s) 10. Gangappa SN, Crocco CD, Johansson H, Datta S, Hettiarachchi C, Holm M, read and approved the final manuscript. Botto JF. The Arabidopsis B-BOX protein BBX25 interacts with HY5, negatively regulating BBX22 expression to suppress seedling Funding photomorphogenesis. Plant Cell. 2013;25(4):1243–57. The work was funded by the National Natural Science Foundation of China 11. Gangappa SN, Holm M, Botto JF. Molecular interactions of BBX24 and (NNSFC, Project grant: 31770721). The funder had no role in study design, BBX25 with HYH, HY5 HOMOLOG, to modulate Arabidopsis seedling data collection and analysis, data interpretation or preparation of the development. Plant Signal Behav. 8(8):e25208–4. manuscript. 12. Qi Q, Gibson A, Fu X, Zheng M, Kuehn R, Wang Y, Wang Y, Navarro S, Morrell JA, Jiang D, et al. Involvement of the N-terminal B-box domain of Availability of data and materials Arabidopsis BBX32 protein in interaction with soybean BBX62 protein. J Biol All data supporting the conclusions of this article are provided within the Chem. 2012;287(37):31482–93. article and its additional files. The genomics sequences data of Moso 13. Lin F, Jiang Y, Li J, Yan T, Fan L, Liang J, Chen ZJ, Xu D, Deng XW. B-BOX bamboo and rice are available in the GigaDB Database (http://gigadb.org/ DOMAIN PROTEIN28 Negatively Regulates Photomorphogenesis by dataset/100498), the Arabidopsis Information Resource, version 10 (TAIR 10) Repressing the Activity of Transcription Factor HY5 and Undergoes COP1- (http://www.arabidopsis.org) and the RGAP 7 database (Rice Genome Mediated Degradation. Plant Cell. 2018;30(9):2006–19. Annotation Project, http://rice.plantbiology.msu.edu/). The gene expression 14. Gangappa SN, Botto JF. The BBX family of plant transcription factors. Trends profile database of NCBI (http://www.ncbi.nlm.nih.gov/geo/), accession: Plant Sci. 2014;19(7):460–70. SRX3282084, SRX3282085, SRX3282086, SRX3282087, SRX3282088, 15. Tiwari SB, Shen Y, Chang HC, Hou Y, Harris A, Ma SF, McPartland M, Hymus SRX3282089, SRX3282090, SRX3282091, SRX3282092, SRX3282093, GJ, Adam L, Marion C, et al. The flowering time regulator CONSTANS is SRX3282094, SRX3282095, SRX3282096, SRX3282097, SRX3282098, recruited to the FLOWERING LOCUS T promoter via a unique cis-element. SRX3282099, SRX3282100, SRX3282101, SRX3282102, SRX3282103, New Phytologist. 2010;187(1):57–66. SRX3282104, SRX3282105, SRX3282106, SRX3282107. 16. Jang S, Marchal V, Panigrahi KC, Wenkel S, Soppe W, Deng XW, Valverde F, Coupland G. Arabidopsis COP1 shapes the temporal pattern of CO Declarations accumulation conferring a photoperiodic flowering response. EMBO J. 2008; 27(8):1277–88. Ethics approval and consent to participate 17. Robson F, Costa MMR, Hepworth SR, Vizir I, Pin˜eiro M, Reeves PH, Putterill J, Not applicable. The plant material used in this study was bamboo plants Coupland G. Functional importance of conserved domains in the flowering- growing in Guilin, Guangxi, and tissue samples of bamboo shoots of time gene CONSTANS demonstrated by analysis of mutant alleles and different heights were obtained. 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